Composition for delivery of hematopoietic growth factor

ABSTRACT

A hematopoietic growth factor delivery composition includes a hematopoietic growth factor, a liquid vehicle, a first biocompatible polymer and a second biocompatible polymer. The composition exhibits reverse-thermal viscosity behavior, due to interaction between the first biocompatible polymer and the liquid vehicle. The second biocompatible polymer helps to protect the first biocompatible polymer from being dissolved in vivo following administration to a host.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims a priority benefit under 35 U.S.C.§119(e) to U.S. Provisional Patent Application No. 60/214,298 entitled“COMPOSITION AND METHOD FOR DELIVERY OF HEMATOPOIETIC GROWTH FACTOR”filed Jun. 26, 2000 and to U.S. Provisional Patent Application No.60/274,891 entitled “COMPOSITION AND METHOD FOR DELIVERY OFHEMATOPOIETIC GROWTH FACTOR” filed Mar. 9, 2001, the entire contents ofeach of which are incorporated herein by reference as if each were setforth herein in full.

FIELD OF THE INVENTION

[0002] The present invention relates to compositions for delivery ofhematopoietic growth factors.

BACKGROUND OF THE INVENTION

[0003] Functionally, hematopoietic growth factors can be considered tobelong to one of three groups. The first or multilineage group includesinterleukin 3 (IL-3) and granulocyte macrophage colony stimulatingfactor (GM-CSF) which act on early colony forming units (CFU's)including colony forming unit-granulocyte, erythrocyte, megakaryocyte,macrophage (CFU-GEMM), colony forming unit-granulocyte-macrophage(CFU-GM), burst forming units erythrocyte (BFU-E) or megakaryocytes,(BFU-MK). The second or unilineage group includes erythropoietin (EPO),granulocyte colony stimulating factor (G-CSF), interleukin 5 (IL-5),macrophage colony stimulating factor (M-CSF) and thrombopoietin (TPO),and act on later hematopoietic progenitors (i.e., colony forming uniterythrocyte (CFU-E), colony forming unit megakaryocyte (CFU-Mk), andcolony forming unit eosinophil (CFU-Eo). The third or “potentiating”group includes interleukin 6 (IL-6), interleukin 11 (IL-11), lymphocyteinhibitory factor (LIF), fibroblast growth factor basic (FGFb), stemcell factor (SCF) and Flt3 ligand (Flt3-L), and act to potentiate theactivities of other hematopoietic factors. Within the third group, SCFand Flt3-L both show marked activity on hematopoietic stem cells andthus have been considered special circumstance/stem cell growth factors.

[0004] G-CSF and GM-CSF are two commonly used hematopoietic growthfactors. The principal action of G-CSF is the stimulation of colonyforming unit granulocyte (CFU-G), which in vivo manifests into anaugmented production of polymorphonuclear leukocyte (neutrophil) as wellas enhancing the phagocytic and cytotoxic functions of neutrophils ingeneral. G-CSF has been shown to be effective in the treatment of severeneutropenia following autologous bone marrow transplantation andhigh-dose chemotherapy. GM-CSF and G-CSF are each used to decrease theperiod of neutropenia seen during this type of therapy and therebyreduces morbidity secondary to bacterial and fungal infections. Whenused as a part of an intensive chemotherapy regimen, G-CSF can decreasethe frequency of both hospitalization for febrile neutropenia andinterruptions in life-saving chemotherapy protocols. G-CSF also hasproven to be effective in the treatment of severe congenitalneutropenias. In patients with cyclic neutropenia, G-CSF therapy, whilenot eliminating the neutropenic cycle, will increase the level ofneutrophils and shorten the length of the cycle sufficiently to preventrecurrent infections. G-CSF therapy can improve neutrophil counts insome patients with myelodysplasia or marrow damage. The neutropenia ofAIDS patients receiving AZT also can be partially or completelyreversed.

[0005] G-CSF is typically administered by subcutaneous injection orintravenous infusion at a dose of 1 to 20 μg/kg per day. Thedistribution and clearance rate from plasma (half-life of 3.5 hours) aresimilar for both routes of administration. A continuous, 24-hourintravenous infusion can be used to produce a steady-state serumconcentration of the growth factor. As with GM-CSF therapy, G-CSF isgiven daily following bone marrow transplantation or intensivechemotherapy will increase granulocyte production and shorten the periodof severe neutropenia. In bone marrow transplantation and intensivechemotherapy patients, continuous daily administration for 14 to 21 daysor longer may be necessary to correct the neutropenia. With lessintensive chemotherapy, fewer than 7 days of treatment may be needed.

[0006] Both G-CSF and GM-CSF will increase the number of marrowprogenitor cells in the circulation, a particularly valuable function inpatients preparing for stem cell collection. Post-transplant infusionsof harvested stem cells together with G-CSF or GM-CSF may reduce theseverity of the post-transplant neutropenia.

[0007] One hematopaetic growth factor that has recently receivedconsiderable attention for its unique properties is Flt3-L. Flt3-L is atransmembrane glycoprotein of approximately 30 kDa. Mouse and humanFlt3-L share significant homology at the amino acid level (˜70%), andshow cross-species reactivity, so testing human Flt3-L in mouse producesthe same or similar biological effects as would occur in the human.Cells known to express Flt3-L include human and mouse T cell lines, aswell as architectural cells of the bone marrow, specifically the bonemarrow fibroblast.

[0008] Some of the myelopoietic, or white blood cell potentiatingeffects attributed to Flt3-L include: 1) an expansion of CD34+ CD38−cell number when used in conjunction with SCF and IL-3; 2) an increasein high proliferative potential colony forming cells (HPP-CFC) andCFU-GM numbers; and 3) in the presence of GM-CSF, the formation of largenumbers of CFU-GM. Individual and direct myelopoietic effects of Flt3-Linclude an increase in CFU-GM, CFU-GEMM and HPP-CFC survival and apreferential induction of macrophages under certain conditions. Flt3-Lalone apparently has minimal or no effects on erythroid andmegakaryocyte progenitors.

[0009] There is substantial data showing that the system of Flt3-L andits receptor also plays an important role in lymphopoiesis, theprocesses involved in normal growth and maturation of lymphocytes. Thisimportant activity has been confirmed in mice made deficient for Flt3-LSystem. In these mice hematopoietic populations are essentially normalbut marked deficiencies of early B cell progenitors are found in thebone marrow. This has led to the suggestion that Flt3-L, perhapsexpressed constitutively by bone marrow fibroblasts, is a normalregulator of B cell lymphopoiesis, while cytokines produced by activatedlymphocytes synergize with Flt3-L in times of stress to accelerate Bcell development.

[0010] In addition to its effects on hematopoietic cells and B cells,Flt3-L has also been shown to stimulate the production of dendriticcells, a highly specialized cell involved in antigen presentation andtherefore, normal immunity. Also, with the observation that Flt3-Lstimulates the production of dendritic cells, Flt3-L has been identifiedfor potential use in the area of vaccines, both traditional delivery ofheat killed or otherwise attenuated agents, as well as protein, peptideor DNA vaccines.

[0011] For additional information on Flt3-L, see, for example, Shurin etal., “FLT3: Receptor and Ligand. Biology and Potential ClinicalApplication”, Cytokine & Growth Factor Reviews, Vol. 9, No. 1, pp.37-48, 1998.

[0012] One of the problems associated with the hematopoietic growthfactors such as G-CSF, GM-CSF, SCF and Flt3-L, is the need for multipledaily injections. This, in turn leads to another common disadvantage ofcurrent injectable therapies such as these, that being the creation of asaw-toothlike effect of plasma drug levels. This is due to the creationof large bolus bursts of drug shortly after injection, leading tosupraphysiologic levels of drug, followed by rapid drops in plasma druglevels as the drug is cleared from the body by normal clearanceprocesses. Upon the next injection, the pattern is repeated with largespikes in plasma levels followed by sub-therapeutic levels until thenext injection. An additional problem with current hematopoietic growthfactor therapy includes fever and mild-to-moderate bone pain in patientsreceiving high doses over a long period. In addition, local skinreactions and mild to moderate splenomegaly have been reported.

[0013] There is a significant need for improved formulations and methodsfor delivery of hematopoietic growth factors that address one or more ofthese problems, especially as treatments involving the use ofhematopoietic growth factors continue to expand.

SUMMARY OF THE INVENTION

[0014] The hematopoietic growth factor delivery composition of thepresent invention provides for sustained delivery of hematopoieticgrowth factors, thereby advantageously increasing the plasma half-lifeof hematopoietic growth factors, and thereby also reducing the number ofadministrations, and therefore the number of injections, required fortreatment. Moreover, the saw-tooth profiles of drug plasma levelsexperienced conventionally should be reduced with less frequentadministrations, as should side effects caused by the frequentinjections with conventional treatments. Furthermore, it has been foundin at least some cases, that the activity of the hematopoietic growthfactor is significantly improved when administered in the composition ofthe present invention, relative to conventional formulation andadministration. Therefore, not only should fewer administrations berequired for a treatment program, but less hematopoietic growth factorshould also be required in many instances, which would be expected togenerally reduce the severity of side effects.

[0015] The hematopoietic growth factor delivery composition of thepresent invention comprises a hematopoietic growth factor, a firstbiocompatible polymer, a second biocompatible polymer and a liquidvehicle. The first biocompatible polymer and the liquid vehicle interactin such a manner and are present in such proportions that thecomposition exhibits reverse-thermal viscosity behavior, in that theviscosity of the composition increases with increasing temperature overat least some temperature range. The second biocompatible polymer is aprotective colloid.

[0016] The reverse-thermal viscosity behavior of the deliverycomposition permits the delivery composition to be administered to ahost as a lower-viscosity flowable medium, which then converts to ahigher-viscosity form in vivo. The hematopoietic growth factor is thenadvantageously released in a sustained manner from the protectiveenvironment of the higher-viscosity form of the delivery composition. Toaccomplish this result, the delivery composition should exhibitreverse-thermal viscosity behavior over at least some temperature rangebelow the physiologic temperature of the host. The presence of thesecond biocompatible polymer helps to protect the composition frompremature degradation in vivo due to invasion by aqueous biologicalfluids, such as are encountered by the delivery composition inside thehost after administration. The inclusion of the second biocompatiblepolymer, therefore, is important to help protect the deliverycomposition so that the delivery composition can successfully make thetransition from the lower-viscosity flowable medium to thehigher-viscosity form following administration. Also, the secondbiocompatible polymer helps to inhibit premature dissolution in vivo ofthe higher-viscosity form, thereby promoting a prolonged release of thehematopoietic growth factor. Surprisingly, the inclusion of the secondbiocompatible polymer has also resulted in an observed significantincrease in the activity of the hematopoietic growth factor under atleast some circumstances. Although the mechanism of this enhancement isnot well understood, the enhancement in activity of the observedhematopoietic growth factor with the composition is significant andsurprising.

[0017] In one embodiment, the composition exhibits a reverse-thermalgelation property, which is a special case of reverse-thermal viscositybehavior in which the higher-viscosity form of the delivery compositionis a gel (i.e., gelatinous substance). In this preferred embodiment ofthe delivery composition, the composition should have a reverse-thermalliquid-gel transition temperature that is no higher than the physiologictemperature of the host. The composition is then administerable to thehost as a flowable medium at a chilled temperature, and as the deliverycomposition warms in the host following administration the deliverycomposition converts to the gel form. Because the gel form is typicallysubstantially immobile, the hematopoietic growth factor is releasedwithin the host at the desired location from the protective environmentof the gel to facilitate sustained delivery of the hematopoietic growthfactor.

[0018] In one preferred embodiment, the delivery composition is in theform of a flowable medium at least at a first temperature and in the gelform at least at a second temperature that is higher than the firsttemperature, but not higher than the physiologic temperature of thehost. For example, when the delivery composition is intended for use bya human host, the first temperature could advantageously be below 20°C., preferably in a range of 10° C. to 20° C., and the secondtemperature could advantageously be in a range of 25° C. to 37° C. Inany event, with a human host the delivery composition should bepreferably in the gel form at 37° C. Also, at the first temperature, thefirst biocompatible polymer is preferably substantially entirelydissolved in the liquid vehicle in the form of a solution that is liquidand flowable to an extent to impart sufficient fluidity to the deliverycomposition so that the delivery composition is administrable to a hostby injection. The hematopoietic growth factor may also be dissolved inthe solvent, or may be in the form of a fine precipitate suspended bythe hematopoietic growth factor/solvent solution. The secondbiocompatible polymer will typically be in the form of a “colloidalsolution” in the liquid vehicle, at least at the first temperature.

[0019] Also, for enhanced performance, the hematopoietic growth factorshould be uniformly dispersed throughout the gel, which can typically beaccomplished by mixing the composition at a temperature at which thefirst biocompatible polymer/liquid vehicle combination is in the form ofa flowable liquid solution of the first biocompatible polymer in theliquid vehicle. In this way the hematopoietic growth factor can bedissolved in or uniformly dispersed throughout the solution, and thenthe temperature of the composition can be raised to convert thecomposition to the gel form for storage prior to use.

[0020] In another aspect, the invention includes a method formanufacturing a composition in which the hematopoietic growth factor isdissolved in or dispersed throughout a solution of the firstbiocompatible polymer and a solvent for the first biocompatible polymer.The solvent is typically an aqueous liquid.

[0021] In yet another aspect, the present invention provides a methodfor packaging and storing the hematopoietic growth factor in theprotective environment of the delivery composition. Handling and storagemay be in a gel or liquid form, as desired.

[0022] Both the foregoing summary description and the following detaileddescription are exemplary and are intended to provide explanation of theinvention as claimed. Other aspects and novel features will be readilyapparent to those skilled in the art from the following detaileddescription of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 is a plot in relation to Example 2 showing pharmacokineticprofiles of G-CSF formulated in buffer (Buffer Formulation) andaccording to one embodiment of the invention (Invention Formulation)described in Table 1. G-CSF in each of the formulations was administeredto Balb/c mice as a single intramuscular (i.m.) injection containing 7μg G-CSF. The concentration of G-CSF in serum was measured via ELISA.Reported are averages of serum G-CSF levels in 4 mice per sampling time.

[0024]FIG. 2 is a timeline of the study design for Example 2 employed toassess the pharmacological action of G-CSF in mice. G-CSF in the BufferFormulation was injected twice daily for 3 days and once the day ofsampling for a total of 7 injections. G-CSF in the Invention Formulationwas administered as a single injection on day 1 of the study withsampling at day 4. Injections were given intramuscularly (i.m.) toBalb-C mice.

[0025]FIG. 3 is a bar graph in relation to Example 2 of thepharmacological action of G-CSF as determined by mobilization of HPP-CFCcells in peripheral blood. G-CSF in the Buffer Formulation was injectedtwice daily for 3 days and once the day of sampling for a total of 7injections. Each injection contained either 0.1, 0.5, or 5.0 ,μg G-CSFfor a total cumulative dose of 0.7, 3.5, or 35 μg G-CSF/mouse,respectively. G-CSF in the Invention Formulation was administered as asingle injection on day 1 of the study with sampling at day 4. Thesingle injection contained either 0.7, 3.5, or 35 μg G-CSF. Injectionswere given intramuscularly (i.m.) in Balb/c mice. Mobilization ofHPP-CFC was determined by quantitating the number of HPP-CFC cells uponcytokine (IL-3)-stimulation of isolated peripheral blood leukocytes.

[0026]FIG. 4 is a plot in relation to Example 4 of the pharmacokineticprofiles of Flt3-L parent, Flt3-L in PGZ-1, and Flt3-L in PGZ-2. Thevarious formulations (described in Table 2) were injectedintramuscularly (i.m.) into Balb/c mice and the levels of Flt3-L inserum were followed for up to 24 hours for the parent formulation orevery 24 hours up to 96 hours for the PGZ formulations.

[0027]FIG. 5A in relation to Example 4 is a bar chart showing whiteblood cell counts (WBC) and FIG. 5B is a bar chart showing spleen cellcounts (SPC) determined in Balb/c mice following intramuscular (i.m.)injection of Vehicle control, Flt3-L parent, Flt3-L in PGZ-1, or Flt3-Lin PGZ-2. Values are Mean±SEM of cell counts determined 96 hoursfollowing administration of the various formulations.

DETAILED DESCRIPTION

[0028] As used herein, “CFU” means colony forming unit, “CFU-GEMM” meanscolony forming unit-granulocyte, erythrocyte, megakaryocyte, macrophage,“CFU-G” means colony forming unit-granulocyte, “CFU-GM” means colonyforming unit-granulocyte-macrophage, “CFU-E” means colony formingunit-erythroid, “CFU-MK” means colony forming unit-megakaryocyte, and“CFU-Eo” means colony forming unit-eosinophil.

[0029] As use herein, “BFU” means burst forming unit, “BFU-E” meansburst forming unit-erythroid, and “BFU-MK” means burst formingunit-megakaryocyte.

[0030] As used herein, “IL” means interleukin.

[0031] As used herein, “EPO” means erythropoietin.

[0032] As used herein “TPO” means thrombopoietin.

[0033] As used herein, “CSF” means colony stimulating factor, “G-CSF”means granulocyte colony stimulating factor, “M-CSF” means macrophagecolony stimulating factor, and “GM-CSF” means granulocyte-macrophagecolony stimulating factor.

[0034] As used herein, “CFC” means colony forming cells and “HPP-CFC”means high proliferative potential colony forming cells.

[0035] As used herein, “FGF” means fibroblast growth factor and ‘FGFb”means fibroblast growth factor basic.

[0036] As used herein, “LIF” means leukocyte inhibitory factor.

[0037] The terms “reverse-thermal viscosity property” and“reverse-thermal viscosity behavior” each refer to a property of amaterial, such as the first biocompatible polymer or the deliverycomposition as the case may be, to undergo a viscosity increase withincreasing temperature across at least some temperature range.

[0038] As used herein, “reverse-thermal gelation property” refers to aproperty of a material, such as the first biocompatible polymer or thedelivery composition, as the case may be, to change from a flowablemedium, typically a liquid form, to a gel form as the temperature israised from below to above a reverse-thermal liquid-gel transitiontemperature.

[0039] As used herein, “reverse-thermal liquid-gel transitiontemperature refers to a temperature at which, or a temperature rangeacross which, a material, such as the first biocompatible polymer or thedelivery composition, as the case may be, changes physical form from aflowable medium, typically a liquid form, to a gel form, as thetemperature of the material is increased.

[0040] The term “reverse-thermal gelation polymer” refers to a polymercapable of interacting with a liquid vehicle so that the polymer/liquidvehicle combination exhibits a reverse-thermal gelation property atleast at some proportions of the polymer and the liquid vehicle.

[0041] As used herein, “biocompatible” means not having toxic orinjurious effects on biological function in a host.

[0042] As used herein, “protective colloid” means a hydrophilic polymerthat has colloidal-size molecules and that is capable of interactingwith water molecules through hydrogen bonding. By “colloidal-size,” itis meant that one or more of the molecular dimensions when dispersed inan aqueous liquid are in a range of one nanometer to one micrometer.

[0043] The present invention provides a composition for delivery ofhematopoietic growth factor to a biologic host, preferably a mammalianhost, and more preferably a human host. The composition comprises atleast one hematopoietic growth factor, at least one liquid vehicle, atleast a first biocompatible polymer and at least a second biocompatiblepolymer that is different than the first biocompatible polymer.Optionally, the composition may also comprise additives such aspenetration enhancers and protective stabilizers, and/or an active agentin addition to the hematopoietic growth factor.

[0044] The hematopoietic growth factor included in the deliverycomposition may be any material capable of stimulating hematopoieticcell activity in the intended host. The delivery composition may includeonly one type of hematopoietic growth factor or may include more thanone different type of hematopoietic growth factors.

[0045] Exemplary hematopoietic growth factors useful in the deliverycomposition of the present invention include those in the multilineagegroup (including IL-3 and GM-CSF), the unilineage group (including EPO,G-CSF, IL-5, M-CSF and TPO) and the “potentiating” group (includingIL-6, IL-11, LIF, FGF b, SCF and Flt3-L).

[0046] The amount of hematopoietic growth factor in the composition ofthe present invention varies depending on the nature and potency of thegrowth factor. In most situations, however, the amount of hematopoieticgrowth factor in the composition will be smaller than about 20% w/wrelative to the first biocompatible polymer.

[0047] The present invention provides a hematopoietic growth factordelivery composition for prolonged, or sustained, delivery ofhematopoietic growth factor, thereby reducing the frequency ofadministrations as part of a treatment. Furthermore, it has been foundthat the delivery system of the present invention, in at least somecircumstances, results in enhanced cell generation relative to the samequantity of hematopoietic growth factor administered by a conventionalmethod. Not to be bound by theory, but to aid in the understanding ofthe invention, it is believed that the composition of the presentinvention reduces or eliminates degradation of the hematopoietic growthfactor and allows for a relatively slow sustained administration ofhematopoietic growth factors to the host. In addition, it is believedthat the composition may be targetting the hematopoietic growth factorto tissues that would make the most efficient use of the hematopoieticgrowth factor.

[0048] The liquid vehicle may be any suitable liquid or mixture ofliquids, but is typically an aqueous liquid. An important aspect of thedelivery composition of the present invention is that the liquid vehicleand the first biocompatible polymer are selected and included in thedelivery composition in such proportions that the delivery compositionexhibits reverse-thermal viscosity behavior over at least sometemperature range. Therefore, the viscosity of the delivery compositionincreases with increasing temperature over some temperature range. At afirst temperature within the temperature range, the delivery compositionis in a lower-viscosity form, in which the delivery composition is inthe form of a flowable medium. At a second temperature in thetemperature range, which second temperature is higher than the firsttemperature, the delivery composition is in a higher-viscosity form thathas a significantly higher-viscosity than the lower-viscosity form.Preferably the viscosity of the higher-viscosity form is at least 1times, more preferably at least 2 times, and even more preferably atleast 3 times as great as the viscosity of the lower-viscosity form.Advantageously, the first temperature is below the physiologictemperature of the host and the second temperature is at or below thephysiologic temperature of the host. In this way, the deliverycomposition is administerable to the host as a flowable medium in thelower-viscosity form at a chilled temperature, with the deliverycomposition converting to the higher-viscosity form as the deliverycomposition warms up inside the host following administration. By“flowable,” it is meant that the delivery composition is sufficientlyfluid so as to be syringable.

[0049] The first biocompatible polymer in the delivery composition ofthe present invention typically is a reverse-thermal gelation polymer.The first biocompatible polymer and the liquid vehicle are selected, andthe delivery composition is formulated with relative proportions of theliquid vehicle and the first biocompatible polymer, so that the deliverycomposition exhibits reverse-thermal viscosity behavior across at leastsome temperature range, preferably a temperature range below 40° C.,more preferably a temperature range below 37° C. and even morepreferably a temperature range within a range of from 10° C. to 37° C.Typically, the delivery composition exhibits reverse-thermal viscositybehavior over at least some temperature range within a range of 1° C. to20° C. Due to the reverse thermal viscosity behavior of the deliverycomposition, the delivery composition can be administered to the host ata cooler temperature where the composition has a lower-viscosity, withthe viscosity of the composition then increasing in the host followingadministration, whereby the mobility of the composition is severelyreduced within the host following administration. In one embodiment, inthe case of a human host, the delivery composition is preferably in theform of the lower-viscosity flowable medium at least at a firsttemperature at or below 20° C., and more preferably in a range of 1° C.to 20° C., and the delivery composition is preferably in thehigher-viscosity form at least at a second temperature in a range offrom 25° C. to 37° C.

[0050] In one preferred embodiment, the liquid vehicle and firstbiocompatible polymer are selected and included in the deliverycomposition in such proportions that the delivery composition has areverse-thermal gelation property, so that the higher-viscosity form isa gel. In this situation, the delivery composition should have areverse-thermal liquid-gel transition temperature that is no higher thanthe physiologic temperature of the host, but that is high enough to beconvenient for administration to the host in the form of a flowablemedium.

[0051] When the delivery composition has a reverse thermal gelationproperty, then the delivery composition will exist in the form of aflowable medium at least at a first temperature and in the form of a gelat least at a second temperature that is higher than the firsttemperature. Preferably both the first and second temperatures are below40° C., and more preferably the second temperature is no higher than 37°C., especially in the case of a human host. A preferred situation iswhen the first temperature is in a range of 1° C. to 20° C. and thesecond temperature is in a range of 25° C. to 37° C.

[0052] Any first biocompatible polymer may be used that, as formulatedin the delivery composition, is capable of interacting with the liquidvehicle to impart the desired reverse-thermal viscosity behavior to thedelivery composition. Non-limiting examples of some reverse-thermalgelation polymers useful for preparing the delivery composition includecertain polyethers (preferably polyoxyalkylene block copolymers withmore preferred polyoxyalkylene block copolymers includingpolyoxyethylene-polyoxypropylene block copolymers referred to herein asPOE-POP block copolymers, such as Pluronic™ F68, Pluronic™ F127,Pluronic™ L121, and Pluronic™ L101, and Tetronic™ T1501); certaincellulosic polymers, such as ethylhydroxyethyl cellulose; and certainpoly (ether-ester) block copolymers (such as those disclosed in U.S.Pat. No.5,702,717). Pluronic™ and Tetronic™ are trademarks of BASFCorporation. Furthermore, more than one of these and/or otherbiocompatible polymers may be included in the delivery composition toprovide the desired characteristics and other polymers and/or otheradditives may also be included in the delivery composition to the extentthe inclusion is not inconsistent with performance requirements of thedelivery composition. Furthermore, these polymers may be mixed withother polymers or other additives, such as sugars, to vary thetransition temperature, typically in aqueous solutions, at whichreverse-thermal gelation occurs.

[0053] Polyoxyalkylene block copolymers are particularly preferred touse as the biocompatible reverse-thermal gelation polymer. Apolyoxyalkylene block copolymer is a polymer including at least oneblock (i.e. polymer segment) of a first polyoxyalkylene and at least oneblock of a second polyoxyalkylene, although other blocks may be presentas well. POE-POP block copolymers are one class of preferredpolyoxyalkylene block copolymers for use as the biocompatiblereverse-thermal gelation polymer in the delivery composition. POE-POPblock copolymers include at least one block of a polyoxyethylene and atleast one block of a polyoxypropylene, although other blocks may bepresent as well. The polyoxyethylene block may be represented by theformula (C₂H₄O)_(b) when b is an integer. The polyoxypropylene block maybe represented by the formula (C₃H₆O)_(a) when a is an integer. Thepolyoxypropylene block could be for example (CH₂CH₂CH₂O)_(a), or couldbe

[0054] Several POE-POP block copolymers are known to exhibitreverse-thermal gelation properties, and these polymers are particularlypreferred for imparting reverse-thermal viscosity behavior properties tothe delivery composition of the present invention. Examples of POE-POPblock copolymers include Pluronic™ F68, Pluronic™ F127, Pluronic™ L121,Pluronic™ L101, and Tetronic™ T1501. Tetronic™ T1501 is one example of aPOE-POP block copolymer having at least one polymer segment in additionto the polyoxyethylene and polyoxypropylene segments. Tetronic™ T1501 isreported by BASF Corporation to be a block copolymer including polymersegments, or blocks, of ethylene oxide, propylene oxide and ethylenediamine.

[0055] As will be appreciated, any number of biocompatible polymers maynow or hereafter exist that are capable of imparting the desiredreverse-thermal viscosity behavior to the delivery composition of thepresent invention, and such polymers are specifically intended to bewithin the scope of the present invention when incorporated into thedelivery composition.

[0056] Some preferred POE-POP block copolymers have the formula:

HO(C₂H₄O)_(b)(C₃H₆O)_(a)(C₂H₄O)_(b)H  I

[0057] which, in the preferred embodiment, has the property of beingliquid at ambient or lower temperatures and existing as a semi-solid gelat mammalian body temperatures wherein a and b are integers in the rangeof 15 to 80 and 50 to 150, respectively. A particularly preferredPOE-POP block copolymer for use with the present invention has thefollowing formula:

HO(CH₂CH₂O)_(b)(CH₂(CH₃)CHO)_(a)(CH₂CH₂O)_(b)H  II

[0058] wherein a and b are integers such that the hydrophobe baserepresented by (CH₂(CH₃)CHO)_(a) has a molecular weight of about 4,000,as determined by hydroxyl number; the polyoxyethylene chain constitutingabout 70 percent of the total number of monomeric units in the moleculeand where the copolymer has an average molecular weight of about 12,600atomic mass units (amu) or Daltons. Pluronic™ F-127, also known aspoloxamer 407, is such a material. In addition, a structurally similarPluronic™ F-68 may also be used.

[0059] The procedures used to prepare aqueous solutions which form gelsof polyoxyalkylene block copolymer are well known and are disclosed inU.S. Pat. No. 5,861,174, which is incorporated herein by reference inits entirety. Typically, the amount of polymer and the amount ofhematopoietic growth factor are selected such that the resultingcomposition has reverse-thermal gelation properties at a transitiontemperature at less than about 37° C., preferably between about 10° C.and 37° C., more preferably between about 20° C. to about 37° C. Theconcentration of the first biocompatible polymer in the composition willvary depending upon the specific first biocompatible polymer and thespecific situation. In most situations, however, the first biocompatiblepolymer will comprise from about 1% by weight to about 70% by weight,and more typically from about 10% by weight to about 33% by weight. Forexample, particularly preferred for Pluronic™ F-127 is a range of fromabout 13% by weight to about 25% by weight.

[0060] The second biocompatible polymer is a protective colloid, and isincluded to impart increased resistance of the delivery composition tophysical deterioration that might otherwise occur then the deliverycomposition encounters extraneous aqueous liquids. This is particularlyimportant to protect the higher-viscosity form, such as the gel, frompremature structural deterioration due to the influence of aqueousbiological fluids following administration to the host. In particular,the first biocompatible polymer is subject to dissolution in aqueousbiological liquids encountered after administration of the deliverycomposition to the host. The second biocompatible polymer helps toinhibit or prevent such dissolution of the first biocompatible polymer,thereby helping to maintain the structural integrity of the deliverycomposition in vivo.

[0061] The second biocompatible polymer is hydrophilic. Preferably, thesecond biocompatible polymer is more hydrophilic than the firstbiocompatible polymer. By having a higher affinity for water than thefirst biocompatible polymer, the second biocompatible polymer tends toprotect the first biocompatible polymer from being dissolved away byaqueous biological fluids present in the host. The protection affordedby the second biocompatible polymer helps to inhibit deterioration ofthe delivery composition, so that the higher-viscosity form of thedelivery composition will endure for some significant time followingadministration, permitting delivery of the hematopoietic growth factorto be sustained over an extended time. Absent the second biocompatiblepolymer, the first biocompatible polymer would be much more susceptibleto dissolution by biological fluids, which could, for example,prematurely destroy integrity of the desired gel character of thehigh-viscosity form of the delivery composition.

[0062] Also, the second biocompatible polymer is of colloidal molecularsize and of high molecular weight. Typically, the weight averagemolecular weight of the second biocompatible polymer is at least 5,000Daltons and more typically, at least 10,000 Daltons. In many situations,the second biocompatible polymer has a weight average molecular weightof at least 50,000 and often 100,000 or more.

[0063] The second biocompatible polymer can be any biocompatible polymerthat acts as a protective colloid in the delivery composition. Thesecond biocompatible polymer will, however, ordinarily be asaccharide-based polymer. By saccharide-based, it is meant that thesecond biocompatible polymer is a polysaccharide or a derivative of apolysaccharide material.

[0064] Cellulosic polymers are particularly preferred for use as thesecond biocompatible polymer, and especially preferred are cellulosicpolymers that are swellable by water. Nonlimiting examples of cellulosicpolymers for use as the second biocompatible polymer includemethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropyl methylcellulose, ethylhydroxyethyl cellulose and carboxymethyl cellulose. A particularlypreferred cellulosic polymer for use as the second biocompatible polymeris hydroxypropyl methylcellulose.

[0065] Also useful as the second biocompatible polymer arephycocolloids. A phycocolloid is a hydrophilic carbohydrate polymeroccurring in algae, and derivatives of such polymers. Some examples ofphycocolloids include carrageenan, algin, and agar. Also useful as thesecond biocompatible polymer are alginates such as, for example, sodiumalginate.

[0066] The hematopoietic growth factor, liquid vehicle, firstbiocompatible polymer and second biocompatible polymer can be present inthe delivery composition in any suitable relative proportions compatiblewith the performance requirements of the delivery composition.Typically, however, the delivery composition will include from0.00000001 weight percent to 0.000005 weight percent of thehematopoietic growth factor, from 60 weight percent to 96 weight percentof the liquid vehicle, from 5 weight percent to 33 weight percent of thefirst biocompatible polymer and from 0. 1 weight percent to 5 weightpercent of the second biocompatible polymer.

[0067] The composition of the present invention can also comprise otheradditives, including polymer or protein stabilizers such as glycerol,trehalose, sucrose, glycine, mannitol, and albumin.

[0068] In one embodiment, the composition may optionally also include anactive agent, in addition to the hematopoietic growth factor, that is tobe delivered to a host along with the hematopoietic growth factor. Inone preferred embodiment, the composition is used for vaccinationpurposes, and the composition includes an antigen in addition to thehematopoietic growth factor. As used herein, antigen refers to anysubstance or material capable of causing an immune response whenadministered to a host. Antigens include, for example, polypeptides,peptides, proteins, glycoproteins and polysaccharides that are obtainedfrom animal, plant, bacterial, viral protozoan and parasitic sources orare produced by synthetic methods, including epitopes of proteins.

[0069] Exemplary antigens which may be included in the composition ofthe present invention include antigens from bacteria, protozoa andviruses against which vaccines are currently available or laterdeveloped, such as antigens from viruses, protozoa or bacteria that arethe causative agents of childhood illnesses, Tetanus toxoid, Diphtheriatoxoid and other non-pathogenic mutants of these toxins, antigens fromBordatella pertussis, antigens from M. tuberculosis, antigens fromP.falciparum, antigens from blood-borne pathogens including Hepatitis Cantigens, and HIV antigens; tumor-specific antigens; and antigensderived from HCG or other molecules involved in the mammalianreproductive cycle. Preferably the antigen is selected from the groupconsisting of tetanus toxoid, diphtheria toxoid and other non-pathogenicmutants of these toxins, other antigens from viruses or bacteria thatare the causative agents of childhood illnesses, antigens from M.tuberculosis, antigens from Bordatella pertussis, antigens from virusesor bacteria against which vaccines are currently available, Hepatitis Cantigens, HIV antigens and antigens from other blood-borne pathogens andtumor-specific antigens. Most preferably the antigen is selected fromthe group consisting of Tetanus toxoid, Diphtheria toxoid and othernon-pathogenic mutants of these toxins, other antigens from viruses orbacteria that are the causative agents of childhood illnesses, antigensfrom M. tuberculosis, antigens from Bordatella pertussis or HIV andantigens from viruses or bacteria against which vaccines are currentlyavailable. Particularly preferred is for the antigen to include one ormore of tetanus toxoid, diphtheria toxoid and antigens from Bordatellapertussis.

[0070] When an antigen is used, the amount of antigen in the compositionof the present invention varies depending on the nature and potency ofthe antigen. Typically, however, the amount of antigen present in thecomposition of the present invention is from about 0.000001% by weightof the composition to about 5% by weight of the composition.

[0071] The composition of the present invention may be administered to ahost to achieve any desired hematopoietic effect. Preferably the host isa mammal, and more preferably a human. The composition can beadministered in a variety of forms adapted to the chosen route ofadministration, e.g., parenterally. Parenteral administration in thisrespect includes administration by the following routes: intravenous,intramuscular, subcutaneous, intrasynovial, transepithelially includingtransdermal, sublingual and buccal, intranasal, and intraperitoneal.Preferably, the mode of administering the composition of the presentinvention is selected from the group consisting of subcutaneous andintramuscular injections, and mucosal routes, including intranasal, withinjection routes being even more preferred.

[0072] The composition is typically prepared in water, a saline solutionor another aqueous liquid as the liquid vehicle. Under ordinaryconditions of storage and use, these preparations can also contain apreservative to prevent the growth of microorganisms. The compositionsuitable for injectable use includes sterile aqueous solutions.Preferably, the composition is sterile with sufficient fluidity for easysyringability. It can be stable under the conditions of manufacture andstorage and preferably preserved against the contaminating action ofmicroorganisms such as bacterial and fungi. The liquid vehicle can be asolvent of dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by maintaining the temperatureof the composition having reverse-thermal gelation properties below thetransition temperature. The prevention of the action of microorganismscan be brought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, e.g., sugars, phosphate buffers, sodium chloride, or mixturesthereof.

[0073] The composition of the present invention can be implanteddirectly into the body by injecting it as a liquid, whereupon thecomposition will ordinarily gel as it warms inside the body when thecomposition has reverse-thermal gelation properties. Also, although thecomposition of the present invention is typically sufficiently fluid andflowable when administered, it will convert to a semi-solid gel insidethe host when it has the proper reverse-thermal gelation properties.Also, the composition can be administered in the form of a gel, forexample by surgical implantation of the gel.

[0074] In another embodiment, solutes, can be incorporated into thedelivery composition of the present invention, for example to stabilizethe hematopoietic growth factor. The use of such protein-stabilizingsolutes, such as, for example, sucrose, not only aid in protecting andstabilizing the protein (i.e., hematopoietic growth factor), but alsoallow the first biocompatible polymer to form suitable gels at lowerconcentrations than needed in water or buffer alone and/or to change thetransition temperature at which reverse-thermal gelation occurs. Thus,the working range of first biocompatible polymer concentration can bewidened and the transition temperature modified. It is known that insome cases a gel will not form when the concentration ofpolyoxyethylene-polyoxypropylene block copolymer in water or dilutebuffer is outside a particular range, e.g., equal to or less than 15%percent by weight for some such polymers. However, by introducingprotein-stabilizing solutes into the composition of the presentinvention, the transition temperature may be manipulated, while alsolowering the concentration of polyoxyethylene-polyoxypropylene blockcopolymer that is necessary to form a gel.

[0075] The hematopoietic growth factor delivery composition can be usedto stimulate hematopoietic cell activity by administering the growthfactor delivery composition to a host by any suitable administrationtechnique. Typically, the delivery composition is chilled at the time ofadministration so as to be in the form of a flowable medium. Also, thefirst biocompatible polymer, and the hematopoietic growth factor, ispreferably substantially entirely dissolved in the liquid vehicle whenadministered to the host.

[0076] In another aspect of the invention, a method is provided forpackaging and storing the hematopoietic growth factor deliverycomposition. According to this aspect of the invention, the deliverycomposition is placed in a container when the composition is in the formof a flowable medium. The temperature of the composition is then raisedso that the delivery composition converts to a gel form within thecontainer for storage. Following storage in the gel form, the deliverycomposition can be converted back to a flowable medium foradministration to the host at the appropriate time by lowering thetemperature of the delivery composition in the container. In this way,the delivery composition is easy to handle during manufacturing andpackaging operations, but can be stored in the highly stable form of agel. Furthermore, the delivery composition can be converted back to aflowable medium for ease of administration. This ability to store thehematopoietic growth factor in the protective gel form prior to use is adistinct advantage with the present invention. Alternatively, thedelivery composition could be stored in the form of a flowable medium ata temperature below the reverse-thermal liquid-gel transitiontemperature, but such a flowable medium is often not as convenient forhandling and storage as a gel form.

[0077] In another aspect, a method for making the delivery compositionis provided, comprising dissolving the first biocompatible polymer in aliquid vehicle and suspending or codissolving the hematopoietic growthfactor and the second biocompatible polymer in the liquid vehicle.Preferably, both the hematopoietic growth factor and the secondbiocompatible polymer are also dissolved in the liquid vehicle duringthe manufacture.

EXAMPLES

[0078] Additional objects, advantages, and novel features of thisinvention will become apparent to those skilled in the art uponexamination of the following examples thereof, which is not intended tobe limiting.

Example 1

[0079] Formulation of G-CSF with Pluronic™ F127

[0080] In one preferred embodiment of the present invention, thehematopoietic growth factor is G-CSF, and the delivery composition ofthe present invention provides a delivery system for the sustainedadministration of G-CSF to a human or animal host. A preferred firstbiocompatible polymer in this situation is a POE-POP block copolymerwith reverse-thermal gelation properties.

[0081] As a specific formulation example, G-CSF can be formulated withPluronic™ F127 (poloxamer 407), with and withouthydroxypropylmethylcellulose (HPMC). Dry powder forms of Pluronic™ F127and HPMC are weighed, mixed together, and then reconstituted in water orphysiological buffer to achieve the drug delivery matrix containing,upon addition of G-CSF, the desired concentrations of each component.More specifically, the concentration of Pluronic™ F127 is one that willachieve a final concentration (e.g., 5-30 weight %) at which it forms asemi-solid gel, along with the addition of HPMC, at body temperature(37° C.). If included, HPMC can be added in an amount to achieve a finalconcentration (e.g., HPMC 0.1-5 weight %) necessary to modulate thephysicochemical or pharmacological properties of the Pluronic™ F127 orG-CSF. Alternatively, Pluronic™ F127 and HPMC can be formulatedseparately as individual solutions and then mixed together to producethe drug delivery matrix containing, upon addition of G-CSF, the desiredconcentrations of each component. As a further alternative, a solutionof either Pluronic™ F127 or HPMC in buffer or water and a dry powder ofthe second polymer (i.e., either Pluronic™ F127 or HPMC) can be mixedtogether to achieve the drug delivery matrix containing, upon additionof G-CSF, the desired concentrations of each component.

[0082] G-CSF can be added to the liquid or dry mixture of Pluronic™ F127and HPMC. The G-CSF can be added in dry powder form, or as a liquidsolution to the drug delivery matrix. Final concentrations of G-CSF inthe Pluronic™ F127 and HPMC drug delivery matrix include thoseconcentrations that will provide biological levels of G-CSF as asustained release following injection. For example, G-CSF can be addedat a concentration so that the injected volume contains dosages of G-CSFthat would provide therapeutic levels for a sustained period afteradministration. More specifically, G-CSF can be incorporated into thedelivery matrix at various desired concentrations, such as for example,to provide 1 to 500 μg/injection of G-CSF.

[0083] The addition of HPMC in the delivery matrix modulates G-CSF insuch a way that the pharmacokinetic profile is altered to providesustained levels of G-CSF in serum compared to G-CSF in only Pluronic™F127. Furthermore, addition of HPMC greatly increases thepharmacological action of G-CSF not only on peripheral bloodhematopoeisis, but also on spleen and bone marrow cell hematopoeisis.Although the mechanism of action of HPMC on the pharmacokinetic profileand pharmacological action of G-CSF is not known, it is proposed thatthe addition of HPMC may 1) stabilize the delivery matrix, 2) stabilizethe hematopoietic growth factor, 3) target the hematopoietic growthfactor to its site of action within the body, and/or 4) enable thehematopoeitic growth factor to exert its hematopoeitic action on earlierprogenitor cells either directly by the growth factor or indirectly bystimulating other cytokines or growth factors.

Example 2

[0084] Administration of G-CSF with Pluronic™ 127

[0085] Formulations including G-CSF, Pluronic™ 127, with and withoutHPMC, are prepared and administered to groups of Balb/c mice todetermine a) the effect of formulating G-CSF in a Pluronic™ 127 and HPMC(Invention Formulation) delivery matrix on the pharmacokinetic profileof G-CSF compared to conventionally (Buffer Formulation) formulatedG-CSF and b) the effects of the Invention Formulation on hematopoieticactivity compared to conventionally formulated G-CSF. The formulationsare administered to mice intramuscularly (i.m.), as a single dose forpharmacokinetic analysis and as either single (for InventionFormulation) or multiple (for Buffer Formulation) doses forhematopoeitic acitivity. The compositions of the formulations are shownin Table 1. TABLE 1 Pluronic ™ F127 (% G-CSF HPMC (% Group w/w) (μg/mL)w/w) Vehicle control, 0 0 0 buffer Vehicle control, gel 17 0 0.1 to 5G-CSF in buffer 0 1 to 300 0 (Buffer Formulation) G-CSF with 17 7 to 1000.1 to 5 Pluronic ™ 127 and HPMC (Invention Formulation)

[0086] Two studies are performed. In the first study, Balb/c micereceive a single i.m. injection of 7 μg G-CSF in either the BufferFormulation or the Invention Formulation. Blood is sampled at varioustimepoints for up to 96 h. G-CSF concentration in serum is determinedvia ELISA. The pharmacokinetic profile of G-CSF from the BufferFormulation and the Invention Formulation are shown in FIG. 1. In thesecond study, a group of mice receive a single i.m. injection of G-CSFin the Invention Formulation at a dose of 0.7, 3.5 or 35 μg/mouse, andanother group of mice receive 7 injections of G-CSF in the BufferFormulation at a dose of 0. 1, 0.5 or 5 μg/injection for a totalcumulative dose of 0.7, 3.5 or 35 μg/mouse. The dosing schedule andstudy design for this second study is outlined in FIG. 2. Thehematopoietic action of these two G-CSF formulations as well as vehiclecontrols was determined by assessing numbers of HPP-CFC cells in theperipheral blood leukocyte fraction 4 days after initiation of G-CSFinjection. HPP-CFC cells were quantitated microscopically afterperforming colony forming assays of cytokine (IL-3)-stimulatedperipheral blood leukocytes. The results of this second study are shownin FIG. 3.

[0087] To summarize the results in the two studies, G-CSF in theInvention Formulation:

[0088] (a) has a longer serum half-life; and

[0089] (b) increases HPP-CFC numbers in peripheral blood better thanconventionally formulated and administered G-CSF.

[0090] The results show a substantial improvement in the delivery ofG-CSF, using the Invention Formulation relative to the BufferFormulation. Referring to FIG. 3, it is particularly surprising andnoteworthy that the Invention Formulation administered in a single 3.5μg of G-CSF injection increased the presence of HPP-CFC in peripheralblood by an amount comparable to the increase produced by 10 times asmuch G-CSF (35 μg ) administered in a multi-injection regimen using theBuffer Formulation. The improvement in delivery of G-CSF can be seen inat least two specific ways: first by providing a more sustainedpharmacokinetic profile (longer half-life) and second as creating anapparently more potent hematopoietic growth factor than is exhibited byconventional formulations. The importance of this can been viewed from avariety of perspectives, including:

[0091] (a) as a component to a marketed hematopoeitic growth factor, itwould likely decrease dose costs of since less growth factor wouldlikely be used to produce the same biological effect;

[0092] (b) from the patient perspective, if a total lower drug dosage isused, it would be expected to produce fewer side effects (which forhematopoeitic growth factors are often dose limiting, including feverand joint pain); and

[0093] (c) also from the patient perspective, a better pharmacokineticprofile leads to fewer injections per course of therapy.

Example 3

[0094] Formulation of Flt3-L with Pluronic™ F127

[0095] In a preferred embodiment of the present invention, thehematopoietic growth factor is Flt3-L, and the pharmaceuticalcomposition of the present invention provides a delivery system for thesustained administration of the Flt3-L to a human or animal. A preferredfirst biocompatible polymer in this situation is a POE-POP blockcopolymer with reverse-thermal gelation properties.

[0096] As a specific formulation example, Flt3-L can be formulated withPluronic™ F127 (poloxamer 407), with or withouthydroxypropylrnethylcellulose (HPMC). Pluronic™ F127 is initiallyformulated in water or physiological buffer at concentrations (e.g.,5-30%) at which it forms a semi-solid gel, along with the addition ofHPMC, at body temperature (37° C.). HPMC may then be added to thePluronic™ F127 formulation at concentrations necessary to modulate thephysicochemical properties of the Pluronic™ F127. (e.g., finalconcentrations of HPMC 1-5%). Alternatively, Pluronic™ F127 and HPMC canbe formulated separately as individual solutions and then mixed togetherto produce the drug delivery matrix containing, upon addition of Flt3-L,the desired concentrations of each component. As a further alternative,dry powder forms of Pluronic™ F127 and HPMC can be mixed together andthen reconstituted in water or physiological buffer to achieve the drugdelivery matrix containing, upon addition of Flt3-L, the desiredconcentrations of each component.

[0097] Flt3-L can be added to the liquid or dry mixture of Pluronic™F127 and HPMC. The Flt3-L can be added in dry powder form, or as aliquid solution to the drug delivery matrix. Final concentrations ofFlt3-L in the Pluronic™ F127 and HPMC drug delivery matrix include thoseconcentrations that will provide biological levels of Flt3-L as asustained release following injection. For example, Flt3-L could beadded at concentrations ranging from about 3 to about 15 μg for proposeddelivery of 1 to 5 μg per day over a 3 day sustained release.

[0098] The addition of HPMC modulates Flt3-L in this deliveryformulation in such a way that although the pharmacokinetic profile ofFlt3-L in serum is not altered compared to a drug delivery matrixcontaining only Pluronic™ F127, the biological activity of Flt3-L onspleen and bone marrow cell differentiation is significantly increased.

Example 4

[0099] Administration of Flt3-L with Pluronic™ 127

[0100] Formulations including Flt3-L, Pluronic™ 127 and, optionally,HPMC are administered to groups of Balb/c mice to determine a) theeffects of the formulations on pharmacokinetics compared toconventionally formulated Flt3-L and b) the effects of the formulationson hematopoietic activity compared to conventionally formulated Flt3-L.The formulations are administered to mice i.m. as a single dose. Thecompositions of the formulations are shown in Table 2. TABLE 2Pluronic ™ Group F127% w/w Flt3-L (μg/mL) HPMC % (w/w) Vehicle control 00 0 (aqueous buffer) Flt-3L parent 0 150 0 (Flt3-L formulated in aqueousbuffer) PGZ-1 22 150 0 (Flt3-L formulated in F127) PGZ-2 22 150 5(Flt3-L formulated in F127/HPMC)

[0101] In a study, mice are sacrificed daily up to 4 days afterinjection and plasma is collected to determine drug pharmacokinetics aswell as circulating and splenic white blood cell counts. Thepharmacokinetic profile of Flt3-L is shown in FIG. 4. FIGS. 5A and 5Bshow circulating white blood cells (WBC) and spleen cell counts (SPC).Particularly significant are the much higher white blood cell and spleencell counts recorded in the case of PGZ-1 vs. PGZ-2, as shown in FIGS.5A and 5B. This is particularly noteworthy because both formulationsexhibit similar pharmacokinetic profiles, as shown in FIG. 4.

[0102] To summarize the results in the above-described studies,formulations made according to the present invention:

[0103] a) have a longer plasma half-life;

[0104] b) increase white cells to a greater extent at equivalent dosescompared to conventionally formulated and administered Flt3-L; and

[0105] c) increase CFU-GM and HPP-CFC in both the bone marrow and spleenbetter than conventionally formulated and administered Flt3-L.

[0106] The results show a substantial improvement in the delivery of theFlt3-L, using the invention including HPMC being exceptionally good. Theimprovement can be seen in at least two specific ways, first byproviding a more sustained pharmacokinetic profile (longer half-life)and secondly as creating an apparently more potent hematopoietic growthfactor than is exhibited by the other formulations. The importance ofthis can been viewed from a variety of perspectives, including:

[0107] a) as a component to a marketed hematopoeitic growth factor, itwould likely decrease dose costs of since less growth factor wouldlikely be used to produce the same biological effect;

[0108] b) from the patient perspective, if a total lower drug dosage isused, it would be expected to produce fewer side effects (which forhematopoeitic growth factors are often dose limiting, including feverand joint pain); and

[0109] C) also from the patient perspective, a better pharmacokineticprofile leads to fewer injections per course of therapy.

[0110] The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter.

What is claimed is:
 1. A hematopoietic growth factor deliverycomposition, the composition comprising: a hematopoietic growth factorcapable of stimulating hematopoietic cell activity when administered toa host; a first biocompatible polymer and a liquid vehicle in which thefirst biocompatible polymer is at least partially soluble at sometemperature, the first biocompatible polymer interacting with the liquidvehicle to impart reverse thermal viscosity behavior to the compositionover at least some temperature range, so that the composition is in alower-viscosity form when the temperature of the composition is at afirst temperature within the range and the composition is in ahigher-viscosity form when the temperature is at second temperaturewithin the range that is higher than the first temperature; and a secondbiocompatible polymer being a protective colloid that inhibits thedissolution into aqueous liquids of the first biocompatible polymer atleast when the composition is in the higher-viscosity form.
 2. Thehematopoietic growth factor delivery composition of claim 1, wherein thefirst temperature is lower than 20° C. and the second temperature ishigher than 25° C.
 3. The hematopoietic growth factor deliverycomposition of claim 2, wherein the first temperature is in a range offrom 1° C. to 20° C. and the second temperature is higher than 25° C. 4.The hematopoietic growth factor delivery composition of claim 3 whereinthe second temperature is 37° C.
 5. The hematopoietic growth factordelivery composition of claim 4, wherein the higher-viscosity form has aviscosity that is at least 3 times as large as the viscosity of thelower-viscosity form.
 6. The hematopoietic growth factor deliverycomposition of claim 1, wherein the lower-viscosity form is a flowablemedium and the higher-viscosity form is a gel.
 7. The hematopoieticgrowth factor delivery composition of claim 2, wherein the secondbiocompatible polymer has an affinity for water such that the secondbiocompatible polymer inhibits deterioration of the gel by invasion ofthe composition by aqueous biologic fluids when the composition isadministered to a biologic host.
 8. The hematopoietic growth factordelivery composition of claim 1, wherein the first biocompatible polymeris a block copolymer.
 9. The hematopoietic growth factor deliverycomposition of claim 8, wherein the block copolymer comprises at leastone block of a polyoxyalkylene.
 10. The hematopoietic growth factordelivery composition of claim 9, wherein the polyoxyalkylene is apolyoxypropylene.
 11. The hematopoietic growth factor deliverycomposition of claim 9, wherein the polyoyxyalkylene is apolyoxyethylene.
 12. The hematopoietic growth factor deliverycomposition of claim 1, wherein the first biocompatible polymer is apolyoxyalkylene block copolymer comprising at least one block of a firstpolyoxyalkylene and at least one block of a second polyoxyalkylene. 13.The hematopoietic growth factor delivery composition of claim 12,wherein the first polyoxyalkylene is a polyoxyethylene and the secondpolyoxyalkylene is a polyoxypropylene.
 14. The hematopoietic growthfactor delivery composition of claim 13, wherein the polyoxyethylenecomprise at least 70 weight percent of the polymer.
 15. Thehematopoietic growth factor delivery composition of claim 12, whereinthe polyoxypropylene has the formula (C₃H₆O)_(b), where b is an integer.16. The hematopoietic growth factor delivery composition of claim 12,wherein the polyoxypropylene has the formula

where b is an integer.
 17. The hematopoietic growth factor deliverycomposition of claim 1, wherein the second biocompatible polymer has aweight average molecular weight of at least 5,000 Daltons.
 18. Thehematopoietic growth factor delivery composition of claim 17, whereinthe second biocompatible polymer comprises a saccharide-based polymer.19. The hematopoietic growth factor delivery composition of claim 17,wherein the second biocompatible polymer comprises a cellulosic polymer.20 The hematopoietic growth factor delivery composition of claim 19,wherein the cellulosic polymer comprises methylcellulose.
 21. Thehematopoietic growth factor delivery composition of claim 19, whereinthe cellulosic polymer comprises hydroxymethylcellulose.
 22. Thehematopoietic growth factor delivery composition of claim 19, whereinthe cellulosic polymer comprises hydroxyethylcellulose.
 23. Thehematopoietic growth factor delivery composition of claim 19, whereinthe cellulosic polymer comprises hydroxypropyl cellulose.
 24. Thehematopoietic growth factor delivery composition of claim 19, whereinthe cellulosic polymer comprises hydroxypropyl methylcellulose.
 25. Thehematopoietic growth factor delivery composition of claim 19, whereinthe cellulosic polymer comprises carboxymethylcellulose.
 26. Thehematopoietic growth factor delivery composition of claim 19, whereinthe second biocompatible polymer comprises ethyl hydroxyethyl cellulose.27. The hematopoietic growth factor delivery vehicle of claim 17,wherein the second biocompatible polymer comprises a secondbiocompatible polymer.
 28. The hematopoietic growth factor deliveryvehicle of claim 17, wherein the second biocompatible polymer comprisesat least one of carrageenan and a derivative of carageenan.
 29. Thehematopoietic growth factor delivery vehicle of claim 17, wherein thesecond biocompatible polymer comprises at least one of algin, alginicacid and an alginate.
 30. The hematopoietic growth factor deliveryvehicle of claim 17, wherein the second biocompatible polymer comprisesan alginate.
 31. The hematopoietic growth factor delivery vehicle ofclaim 17, wherein the pycocolloid comprises agar.
 32. The hematopoieticgrowth factor delivery vehicle of claim 17, wherein the secondbiocompatible polymer comprised a starch.
 33. The hematopoietic growthfactor delivery vehicle of claim 1, wherein the second biocompatiblepolymer has a weight average molecular weight of at least 10,000Daltons.
 34. The hematopoietic growth factor delivery vehicle of claim1, wherein the liquid vehicle is an aqueous liquid.
 35. Thehematopoietic growth factor delivery composition of claim 1, wherein thehematopoietic growth factor comprises EPO.
 36. The hematopoietic growthfactor delivery vehicle of claim 1, wherein hematopoietic growth factorcomprises G-CSF.
 37. The hematopoietic growth factor delivery vehicle ofclaim 1, wherein hematopoietic growth factor comprises IL-5.
 38. Thehematopoietic growth factor delivery vehicle of claim 1, whereinhematopoietic growth factor comprises TPO.
 39. The hematopoietic growthfactor delivery vehicle of claim 1, wherein hematopoietic growth factorcomprises GM-CSF.
 40. The hematopoietic growth factor delivery vehicleof claim 1, wherein hematopoietic growth factor comprises IL-3.
 41. Thehematopoietic growth factor delivery vehicle of claim 1, whereinhematopoietic growth factor comprises IL-6.
 42. The hematopoietic growthfactor delivery vehicle of claim 1, wherein hematopoietic growth factorcomprises IL-11.
 43. The hematopoietic growth factor delivery vehicle ofclaim 1, wherein hematopoietic growth factor comprises LIF.
 44. Thehematopoietic growth factor delivery vehicle of claim 1, whereinhematopoietic growth factor comprises FGF basic. 45 The hematopoieticgrowth factor delivery vehicle of claim 1, wherein hematopoietic growthfactor comprises SCF.
 46. The hematopoietic growth factor deliveryvehicle of claim 1, wherein hematopoietic growth factor comprisesFlt3-L.
 47. The hematopoietic growth factor delivery vehicle of claim 1,comprising an antigen.
 48. The hematopoietic growth factor deliverycomposition of claim 1, wherein the liquid vehicle comprises from 60weight percent to 96 weight percent of the composition, thehematopoietic growth factor comprises from 0.00000001 weight percent to0.000005 weight percent of the composition, the first biocompatiblepolymer comprises from 5 weight percent to 33 weight percent of thecomposition and the second biocompatible polymer comprises from 0.1weight percent to 5 weight percent of the composition.
 49. Thehematopoietic growth factor delivery composition of claim 1, wherein thecomposition is contained within an injection device that is actuatableto administer the composition to the host by injection.
 50. A method ofpackaging and storing the hematopoietic growth factor deliverycomposition of claim 6, comprising placing the composition in acontainer when the composition is in the form of the flowable mediumand, after the placing, raising the temperature of the composition inthe container to convert the composition to the gel for storage, whereinthe gel form in the container can be converted back to the form of aflowable medium for administration to the host by lowering thetemperature of the composition in the container.
 51. The hematopoieticgrowth factor delivery composition of claim 1, wherein the host is amammal.
 52. The hematopoietic growth factor delivery composition ofclaim 1, wherein the host is a human.